The goal of this project is to determine the molecular mechanisms by which gene expression is regulated in a simple eukaryote, the yeast Saccharomyces cerevisiae. In particular, we seek to determine the molecular basis for differentiation of yeast cell type. This proposal is concerned with the following aspects of cell type (mating type) in yeast: (1) The molecular mechanism of action of regulators of gene expression. Our prior work has shown that the yeast mating type locus (MAT) codes for regulator proteins: Alpha2 and al-Alpha2 are negative regulators of gene expression, and Alpha1 is an activator of gene expression. These regulators control expression of a- or Alpha-specific genes at the level of production of RNA. We shall address the following questions: (a) How does the Alpha1 product activate expression of the MFAlpha1 (Alpha-factor) gene? (b) How does the Alpha2 product inhibit expression of the STE6 gene? (c) How does Alpha1-Alpha2 inhibit expression of the HO gene? We shall use a variety of genetic and biochemical methods to address these questions. (2) Mating type interconversion and its regulation. The genetic rearrangement that occurs in mating type interconversion is under a variety of controls. We shall try to determine the molecular basis for several of these controls, some of which concern expression of the HO gene itself. (a) How is expression of the HO gene limited to a specific phase of the cell division cycle? (b) Why do mother cells and not daughter cells express HO? (c) What is responsible for degradation of the HO protein? (d) Why are five genes (SWI1-SWI5) required for expression of HO? (e) Is the HO gene product a component of the HO-dependent endonuclease that appears to be the initiating event in mating type interconversion?